Integration of a copy of the retroviral genome into cellular DNA is necessary for viral replication, can serve as a model for other recombination events, and offers an attractive target for specific antiviral therapy. To mediate integration in vivo, the integrase protein must catalyze two endonuclease reactions. In both cases, the nucleophilic oxygen of an OH group attacks and joins to target DNA. However, the reactions exhibit dramatically different specificities: one uses diverse nucleophiles and a single target site on viral DNA, the other uses one specific nucleophile and almost any target site on host DNA. The objectives of this plan are to learn how integrase interacts with its various substrates and to establish if perturbing these interactions blocks virus replication. The central hypothesis is that the enzyme s central region has distinct sites for viral DNA, host DNA, and the attacking nucleophilic group. This hypothesis is based on the ability of the isolated central domain to catalyze nonspecific alcoholysis (a recently discovered activity that mimics the enzyme s biological actions), which proved that this domain interacts with nucleophiles and nonviral DNA. Moreover, the central region recognizes viral DNA, as revealed by studies of chimeric human immunodeficiency virus type 1/visna virus integrase proteins. The rationale for focusing on substrate recognition sites is that this information will complement structural data to model integration and will identify new antiviral targets. The Specific Aims are to: (1) Understand how integrase interacts with its nucleophilic substrates. The unique perspective of the nonspecific alcoholysis assay will reveal how the structures of the nucleophile or target and the divalent metal cofactor affect these interactions. In addition, the nucleophile site (which is hypothesized to be near the active site in the middle of the central domain) will be identified by examining these interactions using proteins with specific amino acid substitutions. (2) Understand how integrase interacts with its target DNA substrates. Assays of new chimeric integrases will localize the viral DNA site (which is hypothesized to be in the C-terminal portion of the central domain) and the host DNA site (which is hypothesized to be in the N-terminal portion of the central domain). (3) Establish whether interfering with the interactions between integrase and its various substrates is a viable antiviral strategy. Integrase mutations that do not abolish activity but affect nucleophile choice, viral DNA specificity, or target-site selection will be placed into viruses to test the hypothesis that these interactions are rate-limiting for virus replication, i.e., that partial interference will inhibit replication.